番茄花柄脱落相关生长素响应基因ARFs的筛选及其功能的初步鉴定

发布时间：2018-09-04 18:03

【摘要】：生长素作为一种重要的植物激素,它在植物体内的作用不仅与其生物合成密切相关,而且受其信号转导中的转录因子调控。ARF是一类关键的生长素响应因子,近年来其分子机理的研究逐渐成为热点,其在植物各组织器官和多种生理活动中的功能探究取得了很大进展。然而生长素调控植物生长发育是一个极其复杂的过程,存在着严密的调控机制,番茄中ARF家族参与生长素调控作用研究多集中于果实发育与成熟方面,而在花器官脱落方面ARF参与生长素的调控作用机制尚未明确,因此探讨ARF在生长素调控番茄花柄脱落的功能机制具有重要理论和现实价值。本文在此基础上,主要探讨了番茄花柄脱落过程中ARFs家族的表达谱,随后挑选与脱落相关的两个番茄SlARFs基因,获得了其转基因植株,从多个角度深入探究了其在番茄生长发育中的功能,为研究生长素介导的植物生理功能的作用机制奠定了一定的理论基础。主要研究结果如下：1.通过乙烯和IAA处理对番茄花柄外植体脱落率影响的调查,结合离层脱落防御基因Cystein-type endopeptidase的表达信号分析,认为番茄开花后8h是花柄外植体脱落的关键节点,在此之前为生长素抑制脱落时期,而在此以后为乙烯诱导和促进脱落时期。2.采用P5::GUS'ChicoⅢ'转基因番茄的染色信号形象化定位不同时期生长素在花柄离区的分布与强度,并通过横切和纵切扫描观察,在番茄花柄脱落过程中无论是离区还是两端部位,其生长素都在减少,但离区部位的生长素含量始终最高,而维管组织中的含量也较高；皮层和髓部中的含量随脱落进行而快速下降。在第32 h脱落完成后整个花柄的生长素也完全消耗殆尽。不同培养方向对于生长素的运输、分布与消耗影响较大。另外,对脱落过程中IAA含量以及GUS活性变化的检测也验证了8h是花柄外植体脱落的关键节点。3.分别探讨了脱落前期(0-8 h)以及脱落后期(8-32 h)两个阶段中ARFs的表达情况。综合分析发现,在生长素抑制脱落的前期阶段是通过调控ARF1,2,3,4,5,7,8-1,9,,11,12,13,13-1,14和17实现的；而在乙烯诱导脱落的后期作用相反。在前期,生长素介导了ARF8-1,9,11,12,13,13-1和14的表达,乙烯调控了ARF13-1的表达；在后期,ARF14,ARF16和ARF19对乙烯较为敏感。通过后期脱落和未脱落两组的比较分析,筛选出ARF5,6,8,10,11,16以及19被脱落诱导上调,说明这些基因对完成脱落进程起重要作用。另一方面,对番茄花柄外植体中SlARFs基因的绝对表达量分析表明,在正常花柄离区中ARF6-1, ARF8-1, ARF13以及ARF19-1是4个表达量最高的基因,推测其可能对离区发育和分离有更为重要的作用。4.设计出涵盖ARF蛋白N端的保守的DNA结合域(即DBD功能区域)引物,并通过特异引物PCR扩增,分别得到预期435 bp (SIARF2:512～947 bp)和422 bp (SlARF14:562～984 bp)的片段。利用Gateway克隆技术构建了SlARF2及SlARF14的RNAi载体,并通过测序和酶切方法均验证了其正确性,为通过阻断基因的特异性结合来探究目的基因功能提供材料。5.利用改进的“中蔬六号”番茄高效再生体系,采用农杆菌介导遗传转化法将目的载体导入植株,整个过程始终保持0.5 mg/L PPT的抗性筛选压力,并将生根培养基中琼脂含量由原来的0.8%调整为0.7%,有利于在抑菌前提下根系的生长,提高了移栽的成活率。对转化的SlARF2-RNAi PPTR及SlARF14-RNAi PPTR番茄抗性株进行PCR扩增,进一步证明外源基因已成功导转入了番茄基因组中,获得了一部分阳性转化植株,转化率均高于30%。6.对SlARF14的蛋白结构及组织特异性表达进行的分析表明,SlARF14蛋白缺失能够与Aux/IAA异源二聚化结合的CTD区域,SlARF14基因在大部分组织器官中均有表达,尤其是雌蕊,花蕾,叶片以及膨大期的果实中,遗传进化分析表明SlARF14与已被证明与叶片生长发育有关的番茄SlARF10高度同源,推测其可能存在着功能上的相近。对12个Aux/IAAs基因进行表达检测结果显示,大部分Aux/IAAs基因在SlARF14-RNAi与对照植株中的表达无显著差异,暗示对于结构缺失(Ⅲ Ⅳ)的SlARF14,仅有个别Aux/IAAs存在某种形式的调控,相对其它ARFs家族成员,S1ARF14可能具有相对独立的作用。7. SlARF14-RNAi植株叶片边缘更加圆滑卷翘,缺裂变浅或减少,叶色深绿,叶片加厚且侧生叶和小叶间出现一定程度的融合,随叶片生长呈现倾向于横向的扩展膨大的趋势,对叶片发育相关的LeT6和LeEXPA10基因有一定的调控作用；在光合水平的调控上,其在发育早期提高了光合速率,增强了叶绿素积累以及激发了Rubisco酶活的释放,同时上调了RBCL,RBCS和CAB的表达；在激素水平的调控上,其叶片发育涉及了ZR的参与以及IAA的早期调控。8. SlARF14-RNAi植株花器官在发育中易衰败死亡,剥去花瓣及雄蕊,露出子房,发现雌蕊发育不良,柱头出现弯曲,子房发育异常,正常授粉受精受到影响,难以坐果,即便坐果,亦容易形成畸形果实。授粉前后雌蕊和雄蕊中IAA含量普遍偏低,授粉后雌蕊中IAA含量未见显著升高。9. SlARF14-RNAi植株花柄在第8 h,脱落信号尚未完全启动时已有脱落发生,说明前期生长素对其的抑制作用有所减弱；而在后期其脱落速率与对照持平,在第36h全部完成了脱落；在乙烯诱导的脱落过程中,SlARF14-RNAi植株花柄在脱落启动后速率稍慢于对照,表现对乙烯欠敏感。与前文探讨的ARF14基因在脱落不同时期表达模式所反映的结果基本一致。10.对S1ARF2的蛋白结构及组织特异性表达进行的分析表明,S1ARF2具有ARF蛋白完整结构,SIARF2基因在大部分组织器官中均有表达,尤其在根系,花器官以及幼苗中表达较高,遗传进化分析表明S1ARF2与已被证明与叶片衰老及花器官脱落有关的拟南芥AtARF2高度同源,推测其可能存在着功能上的相近。对12个Aux/IAAs基因进行表达检测结果显示,大部分Aux/IAAs基因在SlARF2-RNAi与对照植株中的表达无显著差异,S1ARF2的功能调控可能是通过与IAA1,IAA3,IAA7以及IAA26等发生特异性相互作用来进行的。11.通过对PrOARF2:GUS的幼苗染色检测SIARF2在不同发育时期植株体内不同部位的分布情况发现,SIARF2基因主要集中新生叶基部、生长点、叶腋、一级主茎部位(尤其是表皮毛)、侧根基部、须根发生部位以及完全开放的花的雄蕊中强烈表达。证明S1ARF2集中在幼嫩组织或生长发育活跃部位表达,影响雄蕊成熟。12. SIARF2-RNAi植株叶片叶色鲜绿,侧生叶间小叶数量减少。在衰老过程中,与对照植株叶片在老化时大规模快速萎黄死亡相比,SlARF2-RNAi植株叶片延缓了老化进程,作为衰老上调基因,SENU1和SENU5基因的延迟表达证明了这一点；在光合水平上,同一时期其叶片可维持相对较高的存活率,个体叶片光合速率,最大光化学效率,叶绿素残留量,Rubisco酶活性下降均慢于对照,同时RBCL,RBCS基因的下调相对滞后；抗氧化酶活性下降速率大大慢于对照；在激素水平上,其叶片衰老独立于ZR途径,涉及IAA的调控。13.SIARF2-RNAi植株花器官的花梗及萼片部位的表皮毛明显减少,花器官(包括萼片,花瓣,雄蕊,雌蕊)长度均小于对照。授粉前后雌蕊和雄蕊中IAA含量普遍偏低,其雄蕊对于IAA的敏感性有所降低。14. SIARF2-RNAi植株花器官在自然脱落的第28 h以前均小于对照,而关键时期在16～20h之间,这期间脱落率上升非常缓慢,脱落有所延迟,而在20 h以后恢复正常；在乙烯诱导的脱落过程中,SlARF2-RNAi植株花柄脱落率仅在8h时处于极低水平,这时虽在乙烯诱导下脱落亦难以发生,其他时间点与对照近乎一致。
[Abstract]:As an important plant hormone, auxin is not only closely related to its biosynthesis, but also regulated by transcription factors in its signal transduction. ARF is a kind of key auxin-responsive factors. In recent years, the study of its molecular mechanism has gradually become a hot spot in plant tissues and organs and a variety of physiological activities. However, auxin regulation of plant growth and development is a very complex process, and there is a rigorous regulatory mechanism. Research on the role of ARF family in the regulation of auxin in tomato mainly focuses on fruit development and maturation, while in the aspect of flower organ abscission, ARF is involved in the regulation mechanism of auxin. Therefore, it is of great theoretical and practical value to study the functional mechanism of ARF in auxin regulation of petiole abscission in tomato. On this basis, the expression profiles of ARFs family in the process of petiole abscission in tomato were mainly discussed. Then two genes related to abscission were selected and transgenic plants were obtained. The main results are as follows: 1. Investigation on the effect of ethylene and IAA treatment on the explant abscission rate of tomato flower stalk, combined with the abscission defense gene Cystein-type endopeptida. Se expression signal analysis showed that 8 h after flowering was the key node of explant abscission, before which was the period of auxin inhibition abscission, and after that was the period of ethylene induction and promotion abscission. The content of auxin in the detached area and the two ends of the petiole was decreased, but the content of auxin in the detached area was always the highest, and that in the vascular tissue was also higher. The content of cortex and pith decreased rapidly with the detached process. In addition, the changes of IAA content and GUS activity in the process of abscission also confirmed that 8 h was the key node for the abscission of petiole explants. 3. The early abscission stage (0-8 h) and the late abscission stage (8-3) were discussed respectively. 2 h) The expression of ARFs in the two stages. It was found that at the early stage of auxin inhibiting abscission, ARF1, 2, 3, 4, 5, 7, 8-1, 9, 11, 12, 13, 13-1, 14 and 17 were regulated by regulating the expression of ARF13-1, 9, 11, 12, 13-1 and 14, while at the later stage of ethylene-induced abscission, auxin mediated the expression of ARF8-1, 9, 11, 12, 13-1 and 14. The results showed that ARF5, 6, 8, 10, 11, 16 and 19 were up-regulated by abscission induction, indicating that these genes played an important role in the process of abscission. The results showed that ARF6-1, ARF8-1, ARF13 and ARF19-1 were the four most expressed genes in the normal petiole exfoliation region. It was speculated that ARF6-1, ARF13 and ARF19-1 might play a more important role in the exfoliation development and isolation. 4. The primers encompassing the conserved DNA binding domain (that is, the DBD functional region) of the N-terminal of ARF protein were designed and amplified by PCR with specific primers. 5 BP (SIARF2:512-947 bp) and 422 BP (SlARF14:562-984 bp) fragments were constructed by Gateway cloning technique. The RNAi vectors of SlARF2 and SlARF14 were sequenced and digested to verify their correctness, which provided materials for exploring the function of the target gene by blocking the specific binding of genes. 5. The improved "Zhongshu VI" was used. "Tomato high-efficiency regeneration system, using Agrobacterium tumefaciens-mediated genetic transformation method to introduce the target vector into plants, the whole process has always maintained 0.5 mg/L PPT resistance screening pressure, and the agar content in rooting medium from 0.8% to 0.7%, is conducive to the growth of roots under the premise of bacteriostasis, improve the survival rate of transplantation. SlARF2-RNAi PPTR and SlARF14-RNAi PPTR resistant tomato plants were amplified by PCR. It was further proved that foreign genes had been successfully transfected into tomato genome, and some positive transgenic plants were obtained. The transformation rate was higher than 30%. 6. The analysis of SlARF14 protein structure and tissue-specific expression showed that the deletion of SlARF14 protein could be associated with Aux/I. SlARF14 gene was expressed in most tissues and organs, especially in pistils, buds, leaves and fruits during enlargement. Genetic evolution analysis showed that SlARF14 was highly homologous to SlARF10, which had been proved to be related to leaf growth and development. It was speculated that there might be functional similarities between SlARF14 gene and SlARF10 in 12 tomatoes. The results of Aux/IAAs gene expression test showed that most of the Aux/IAAs gene expression in SlARF14-RNAi was not significantly different from that in the control plants, suggesting that only a few Aux/IAAs had some form of regulation on SLARF14 with structural deletion (III IV). Compared with other ARFs family members, S1ARF14 may have a relatively independent role. The edge of leaves of RNAi plants was more smooth and curly, the absence of fission was lighter or less, the color of leaves was dark green, the leaves were thickened and the lateral leaves and interlobular fusion appeared to a certain extent. With the growth of leaves tended to expand laterally, the genes of LeT6 and LeEXPA10 related to leaf development were regulated to some extent at photosynthetic level. On the other hand, it increased the photosynthetic rate, enhanced the accumulation of chlorophyll and stimulated the release of Rubisco enzyme activity, and up-regulated the expression of RBCL, RBCS and CAB. On the regulation of hormone level, the leaf development involved the participation of ZR and the early regulation of IAA. It was found that the pistil was undeveloped, the stigma was curved, the ovary was abnormal, normal pollination and fertilization were affected, it was difficult to set fruit, even fruit setting, it was easy to form deformed fruit. The petiole shedding occurred at the 8th hour before the shedding signal was fully activated, indicating that the inhibitory effect of auxin on the petiole shedding was weakened, and the shedding rate was the same as that of the control at the later stage, and all the petioles shedding was completed at the 36th hour. In the process of ethylene-induced shedding, the petiole shedding rate of SlARF14-RNAi plants was slightly slower than that of the control. The results showed that S1ARF2 had an intact structure of ARF protein and SIARF2 gene was expressed in most tissues and organs, especially in roots and floral organs. Genetic evolution analysis showed that S1ARF2 was highly homologous to Arabidopsis AtARF2, which had been proved to be associated with leaf senescence and floral organ abscission. There was no significant difference in the expression of SIARF2. The function of S1ARF2 may be regulated by specific interaction with IAA1, IAA3, IAA7 and IAA26. 11. The distribution of SIARF2 in different parts of plants at different developmental stages was detected by PrOARF2: GUS seedling staining. Strong expression of S1ARF2 in axillary, primary stem (especially epidermal hairs), lateral root base, fibrous roots and stamens of fully open flowers showed that S1ARF2 was concentrated in young tissues or active parts of growth and development, affecting stamen maturation. Compared with the control plants, the leaves of SlARF2-RNAi plants delayed the aging process, which was proved by the delayed expression of SENU1 and SENU5 genes as the up-regulated genes of senescence. The decreases of photochemical efficiency, chlorophyll residue and Rubisco enzyme activity were slower than those of the control, while the decreases of RBCL and RBCS genes were relatively lagged; the decreases of antioxidant enzyme activity were much slower than those of the control; at the hormone level, leaf senescence was independent of ZR pathway and involved in IAA regulation. 13. SIARF2-RNAi plant pedicels and sepals of floral organs. The length of flower organs (including sepals, petals, stamens and pistils) was less than that of the control. The content of IAA in pistils and stamens was generally low before and after pollination, and the sensitivity of stamens to IAA was decreased. 14. The flower organs of SIARF2-RNAi plants were less than that of the control before 28 h of natural abscission, and the critical period was between 16 and 20 h. In the process of ethylene-induced abscission, the rate of petiole abscission of SlARF2-RNAi plants was only at a very low level at 8 h, which was difficult to occur even under ethylene-induced abscission, and the other time points were almost the same as the control.
【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S641.2

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